1. Field of the Invention
The present invention relates to the fields of molecular biology and drug discovery. In particular, the invention relates to a proton channel protein, nucleic acids encoding the protein, cells engineered to express the protein, assays for compounds affecting the activity of the protein, and the use of such compounds in the treatment of diseases and disorders.
2. Description of the Related Art
Voltage-dependent proton (H+) conductances (GvH+) were first discovered in molluscan neurons (Thomas et al. (1982), Nature 299: 826-8) and later identified in a variety of mammalian cell types such as: alveolar epithelial cells (DeCoursey (1991), Biophys. J. 60:1243-53), macrophages (Kapus et al. (1993) J. Gen. Physiol. 102:729-760), skeletal muscle (Krause et al. (1993), Neuromuscul. Disord. 3:407-11), osteoclasts (Nordstrom et al. (1995), J. Biol. Chem. 270: 2203-12), microglia (Eder and DeCoursey (2001), Prog. Neurobiol. 64:277-305), lymphocytes (Schilling et al. (2002) J. Physiol. 545:93-105), and others (reviewed in DeCoursey (2003), Physiol. Rev. 83:475-579). Indirect evidence suggests that GvH+ are expressed in mammalian hippocampal neurons (Sheldon and Church (2002), J. Neurophysiol. 87:2209-24; Diarra et al. (1999), Neuroscience 93:1003-16). Among cells that have been tested, the highest density of voltage-dependent proton current is found in phagocytic leukocytes of the innate immune system (neutrophils and eosinophils) (DeCoursey (2003), supra).
Clearance of microbial, fungal and parasitic infections by phagocytes requires nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, as evidenced by the development of chronic granulomatous disease (CGD) in humans and mice lacking functional gp91Pphox, the electron-transporting transmembrane subunit of the NADPH oxidase complex (Smith and Curnutte (1991), Blood 77:673-86). Activation of professional phagocytes (i.e. by bacterial peptides or complement) leads gp91phox-dependent secretion of superoxide anion (O2.−) and concomitant generation of intracellular protons (H+) and an outward electron (e−) current (Henderson et al. (1987), Biochem. J. 246:325-9). Henderson and colleagues first postulated that a proton conductance could serve a charge-compensating role to limit intracellular acidification and thereby sustain O2.− production (Henderson et al. (1987), supra); this hypothesis was extended and refined by DeCoursey and colleagues (DeCoursey (2003), Physiol. Rev. 83:475-579; Murphy and DeCoursey (2006), Biochim. Biophys. Acta 1757(8):996-1011).
The core biophysical features of GvH+ elucidated using patch-clamp electrophysiology are: 1) Activation of H+ conductance bye depolarizing (positive) voltage; 2) Sensitivity to the transmembrane [H+] (i.e. pH) gradient, which results in a shift of the threshold for voltage-dependent activation; 3) H+-selective permeation (i. Na+, K+, and Cl− ions do not contribute to the measured current); 4) relatively slow activation kinetics (100's of msec time constants) and faster deactivation kinetics (tens of msec time constants)(DeCoursey (2003), supra). Under steady-state conditions in intact cells, these features dictate that GvH+ are manifested as and outwardly-rectifying H+ currents that result in net H+ extrusion from cells and consequent intracellular alkalinization. The existence of a protein that would generate ionic currents with the properties of GvH+ was long postulated but no cDNA sufficient to unambiguously reconstitute GvH+ was reported until 2006 (Sasaki et al. (2006), Science 312:589-92; Ramsey et al. (2006), Nature 440:1213-6).
Voltage-dependent cation channels share an archetypal structure composed of two distinct domains: the VSD and the pore (P) domain (Long et al (2005a), Science 309(5736):897-903). The P domain is responsible for imparting cation-selective permeation whereas the VSD translates changes in the transmembrane electrical potential into protein conformational changes that lead to channel gating (Long et al. (2005b), Science 309(5736):903-8; Jiang et al. (2003), Nature 423:33-41).